The enzyme tyrosine hydroxylase catalyzes the rate-limiting step in the biosynthesis of catecholamine neurotransmitters. The major aim of the experiments described in this proposal is to characterize the following three biochemical events which rapidly modify tyrosine hydroxylase activity by changing the affinity of the enzyme for its pterin cofactor: 1) anionic phospholipids, 2) enzymatic phosphorylating conditions, and 3) partial proteolysis. Each of these events can activate tyrosine hydroxylase; a comparative study will yield information concerning whether or not the activations occur via a common or through distinct mechanisms. The extension of a current purification procedure should produce homogeneous tyrosine hydroxylase from rabbit adrenal glands. Pure enzyme preparations are required to conclude our study on the mechanism of activation of the hydroxylase by enzymatic phosphorylation. Two models remain to be tested: the catalytic region of the hydroxylase may be directly phosphorylated or a regulatory subunit may be phosphorylated and subsequently dissociate from the enzyme. One class of anionic phospholipids, the phosphoinositides, have the remarkable ability to either stimulate or inhibit tyrosine hydroxylase, depending upon preincubation conditions. The phosphoinositides comprise a very small portion of the total phospholipid in nervous tissue, but they are the only class of phospholipids which turn over rapidly in response to cellular stimuli, e.g. depolarization. Experiments have been designed to characterize the mechanism(s) of how the phosphoinositides may modulate tyrosine hydroxylase activity, using both purified enzyme preparations and synaptosomal or neuroblastoma cell culture preparations where in situ tyrosine hydroxlation may be measured. In the course of the purification of tyrosine hydroxylase, a factor has been isolated which prevents the phosphatidylinositol-induced inactivation of this enzyme. Experiments have been designed to isolate and characterize this potentially physiologically important endogenous modulator of tyrosine hydroxylase activity.